The statements in this section merely provide background information related to the present disclosure and may not constitute prior art.
When a two-photon excited fluorescence (TPEF) microscope is used to image deep tissue, out-of-focus background can arise from both ballistic and non-ballistic excitation. TPEF microscopy has become a well-established tool for high-resolution imaging in scattering media such as thick tissue. While it is well accepted that TPEF microscopy provides greater imaging depth penetration in thick tissue than more conventional fluorescence imaging techniques, such as confocal or widefield microscopy, the depth penetration of TPEF microscopy remains nonetheless limited. For example, demonstrations of TPEF imaging beyond 500 pm in brain tissue have been rare.
Several factors limit TPEF microscopy depth penetration in thick tissue, three of which are described below for exemplary purposes:
1) An excitation beam can undergo scattering when it propagates through tissue. This scattering weakens the ballistic (un-scattered) excitation power that attains the beam focus and thereby reduces the TPEF signal generated at the focus. Since scattering scales roughly exponentially with propagation distance, by dint of the Lambert-Beer law, the reduction in TPEF signal becomes particularly severe at larger focal depths. One strategy to maintain adequate ballistic excitation power at relatively large focal depths has involved the use of non-standard laser sources based on regenerative amplifiers. Unfortunately, such a strategy can only go so far in compensating for an exponential loss in ballistic power, even though it has been the most successful to date in pushing the limits of depth penetration.
2) The required increase in excitation power necessary to maintain (or try to maintain) adequate ballistic power at the beam focus can lead to significant power densities near the tissue surface. If the tissue is fluorescent near its surface, as is the case for example if the fluorescent labeling is homogeneously distributed throughout the sample, or if the sample is autofluorescent either intrinsically or due to superficial tissue damage, then the power density of the ballistic light near the surface can be so high as to produce out-of-focus background fluorescence that is non-negligible compared to the in-focus signal fluorescence. When this background fluorescence begins to dominate signal fluorescence, there is no point in attempting to image deeper in the tissue.
3) At depths where the scattered light is so strong and the ballistic light so weak that the power density of the ballistic light cannot compete with that of the scattered light near the beam focus, then again there is no point in attempting to image deeper in the tissue. Inasmuch as scattering in biological tissue is very dominantly forward directed, the scattered light that exhibits the greatest power density is the light that is only slightly deviated from its ballistic path, as can be verified by Monte-Carlo simulation. Light paths that are only slightly deviated are often referred to as “snakelike”, as opposed to the more severely scattered “diffusive” paths. Snakelike scattering leads to a blurred halo of background fluorescence surrounding the in-focus signal fluorescence. While at shallow depths, this background halo is usually negligible compared to the signal, at larger depths the background halo can become quite problematic.
Thus, a heretofore unaddressed need exists in the industry to increase depth penetration of TPEF microscopy and to address the aforementioned deficiencies and inadequacies.